Myocardial infarction (MI) (irreversible damage to heart tissue, often due to heart attack) is a common life-threatening event that may cause sudden death or heart failure. Normal adaptive mechanisms in response to myocardial infarction (MI) commence with scar formation in the damaged wall progressing to hypertrophy of the unaffected regions which ultimately succumbs to ventricular dilation and heart failure (Malki, Q., Clinical Presentation, hospital length of stay, and readmission rate in patients with heart failure with preserved and decreased left ventricular systolic function, Clin. Cardiol. 25:149-152, 2002). In most cases, the loss of cardiomyocytes after myocardial infarction is irreversible. Despite considerable advances in the diagnosis and treatment of heart disease, cardiac damage and dysfunction relating to myocardial infarction are still among the major cardiovascular disorders. Accordingly, it remains a major therapeutic challenge to find new effective approaches to improve cardiac function after myocardial infarction.
Stem cell therapy to restore infarcted myocardium has been extensively studied, with numerous reports that hematopoietic (Orlic et al., Bone marrow cells regenerate infarcted myocardium, Nature 410:701-5, 2001; Jackson et al., Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells, J. Clin. Invest. 107:1395-402, 2001), and mesenchymal (Mangei et al., Mesenchymal stem cells modified with Aid prevent remodeling and restore performance of infarcted hearts, Nat. Med. 9:1195-201, 2003) stem cells derived from bone marrow (BMCs) can give rise to new myocardium via transdifferentiation. This in turn has rapidly translated into a whirlwind of clinical activity aimed at duplicating these effects in the human heart (Strauer et al., Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans, Circulation 106:1913-1918, 2002; Assmus et al., Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI), Circulation 106: 3009-3017, 2002; Perin et al., Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure, Circulation 107: 2294-2302, 2003; Tse et al., Angiogenesis in ischaemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation, Lancet 361: 47-49, 2003). However, three recent studies (Balsam et al., Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium, Nature 428: 668-73, 2004; Murry et al., Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts, Nature 428: 664-8, 2004; Nygren et al., Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation, Nat. Med. 10: 494-501, 2004) have rigorously challenged the conclusions of these reports by independently demonstrating that BMCs transplanted into damaged hearts could not give rise to cardiomyocytes. Balsam et al. (Balsam et al., Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium, Nature 428: 668-73, 2004) have shown that not only do BMCs fail to give rise to cardiomyocytes, they actually develop into different blood cell types, despite being in the heart. The beneficial effects noted in earlier studies in terms of ventricular performance might be partially attributable to angioblast-mediated vasculogenesis (Kocher et al., Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function, Nat. Med. 7:430-6, 2001) which could prevent apoptosis of native cardiomyocytes rather than by direct myogenesis.
Given these limitations in BMCs, the search for naturally occurring, authentic heart progenitor cells has begun in earnest, with several groups having reported on the existence of such cells (Matsuura et al., Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes, J. Biol. Chem. 279:11384-91, 2004; Oh et al., Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction, Proc. Natl. Acad. Sci. USA 100:12313-8, 2003; Beltrami et al., Adult cardiac stem cells are multipotent and support myocardial regeneration, Cell 114:763-76, 2003; Martin et al., Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac side-population cells in the developing and adult heart, Dev. Biol. 265: 262-75, 2004; Laugwitz et al., Postnatal isl1+cardioblasts enter fully differentiated cardiomyocyte lineages, Nature 433: 647-53, 2005). These native progenitors are alone clearly inadequate in reversing the downward spiral of events culminating in heart failure. Their differentiation in response to environmental cues might be expected to generate cardiomyocytes of a postmitotic nature, hence limiting the ability of such endogenous processes to counter the massive myocyte death in MI.
It is widely accepted that the proliferative (and, therefore, the regenerative) potential of adult mammalian cardiomyocytes is quite limited (Rumyantsev and Carlson, Growth and Hyperplasia of Cardiac Muscle Cells (New York: Harwood Academic Publishers, 1991)), although this view has recently been challenged (Leri et al., Mol. Cell. Cardiol., 3:385-90, 2000; Kajstura et al., Am. J. Pathol., 156:813-19, 2000; Beltrami et al., N. Engl. J. Med., 344(23):1750-57, 2001).
The potential to reactivate cardiomyocyte proliferation through the manipulation of putative cellular regulators, or the conversion of pluripotent stem cells to cardiomyocytes (Orlic et al., Nature, 410:701-05, 2001), offers an exciting impetus for the design of novel therapeutic interventions to enhance cardiac function during disease conditions. The bulk of evidence obtained over the past decade maintains, however, that mammalian cardiomyocytes proliferate throughout fetal development and into the early neonatal period, at which time DNA replication declines quickly and cell division ceases (Beinlich and Morgan, Mol. Cell. Biochem., 119:3-9, 1993; Casscells et al., J. Clin. Invest., 85:433-41, 1990; Speir et al., Circ. Res., 71:251-59, 1992; Parker and Schneider, Annu. Rev. Physiol., 53:179-200, 1991; Simpson, P. C., Annu. Rev. Physiol., 51:189-202, 1989).
Transition from hyperplastic growth (cell division) to hypertrophic growth (increase in cell size) then ensues. In the murine heart, cardiomyocyte division is reportedly completed by birth, with DNA synthesis in neonatal cells (through post-natal day 3) contributing only to binucleation (Soonpaa et al., J. Mol. Cell. Cardiol., 28:1737-46, 1996). The cessation of myocyte proliferation is attributed to an arrest of the cell cycle (Brooks et al., Cardiovasc. Res., 39:301-11, 1998). In accordance with this hypothesis, adult rat cardiomyocytes have been shown to display a dual cell-cycle blockade, with approximately 80% of cells arresting in GO/G1, and 15%-20% of cells arresting in G2/M (Poohnan and Brooks, Mol. Cell. Cardiol., 29:A19 (Abstract), 1997; Poolman et al., Int. J. Cardiol., 67:133-42, 1998).
Progression through the cell cycle is tightly regulated, and involves cyclins complexed with their catalytic partners, the cyclin-dependent kinases (cdks). Among the cyclins, cyclin A2 is unique in that it regulates progression through two critical transitions: cyclin A2 complexed with cdk2 is essential for the G1/S transition, and cyclin A2 complexed with cdk1 promotes entry into mitosis (Sherr and Roberts, Genes Dev., 9:1149-63, 1995; Pagano et al., EMBO J., 11:961-71, 1992). It is well established that mammalian cardiomyocytes cease to proliferate in the early neonatal period due to arrest of the cell cycle. Cyclin A2 is the only cyclin to be completely downregulated, at both the message and protein level, during cardiogenesis, in rats and humans, in a manner that appears coincident with this withdrawal of cardiomyocytes from the cell cycle (Yoshizumi et al., J. Clin. Invest., 95:227580, 1995).
Previously, it has been shown that zebrafish fully regenerate hearts within 2 months of 20% ventricular resection, due to robust proliferation of cardiomyocytes localized at the leading epicardial edge of the new myocardium. This injury-induced cardiomyocyte proliferation was able to overcome scar formation, allowing cardiac muscle regeneration. It has been suggested that this regeneration of heart tissue in zebrafish is related to the Mpsl 30 mitotic checkpoint kinase (Poss et al., Heart regeneration in zebrafish, Science, 298:2188-90, 2002). It has also been shown that cardiomyocytes react to myocardial infarction by activating cyclins and cyclin-dependent kinases (Reiss et al., Myocardial infarction is coupled with activation of cyclins and cyclin-dependent kinases in myocytes, Exp. Cell Res., 225:44-54, 1996).
The inventors of the present application have recently shown that regulation of cyclins, particularly cyclin A2, can induce cardiomyocyte mitosis once the timeline for cell-cycle exit (and, therefore, “terminal” differentiation) has been surpassed. The inventors have now, for the first time, difinitively demonstrated that significant myogenesis can be achieved in mammalian infarcts with sustained recovery of cardiac function. As described herein, the inventors have provided a novel model system in which mice constitutively expressing cyclin A2 in the myocardium elicit a regenerative response after infarction and exhibit limited ventricular dilation and heart failure. The inventors discovered new cardomyocyte formation in the infarcted zones as well as cell cycle re-entry of peri-infarct myocardium with an increase in DNA synthesis and mitotic indices. This enhanced cardiac function was serially assessed by magnetic resonance imaging. The inventors further demonstrated that the constitutive expression of cyclin A2 augments endogenous regenerative mechanisms via induction of side-population cells with enhanced proliferative capacity. The ability of cultured transgenic cardiomyocytes to undergo cytokinesis provides mechanistic support for the regenerative capacity of cyclin A2.